Intestinal dendritic cells, gatekeepers preventing ethanol-induced liver disease
Authors: Cristina Llorente, Wiramon Rungratanawanich, Suthat Liangpunsakul
Journal: Hepatology (2023)

Alcohol-associated liver disease (ALD) is a global illness with high morbidity and mortality, accounting for 5.3% of all deaths worldwide. The lack of treatments other than alcohol abstinence evidences the necessity of research in the field.1 Novel effective therapies along with the mechanism of ALD pathogenesis must be determined to mitigate the global burden of this devastating disease. A delicate interplay between the gut and liver is crucial in the pathophysiology of ALD. Ethanol misuse abruptly disrupts the components shielding the gut-liver balance. Ethanol promotes gut dysbiosis, small intestinal bacterial overgrowth, intestinal permeability, and microbial translocation to the circulation and liver, which triggers inflammation and aggravates ALD.2 Understanding the critical factors of this complex equation is essential for ALD prevention and therapy. The host intestinal immune system influences the intestinal microbiota abundance and composition and participates in the regulation of the gut-liver axis. Understanding the implications and mechanistic pathways of the intestinal immune system in ALD pathogenesis is of relevance, as it may lead to the identification of targeted therapies. Dendritic cells (DCs) are bone marrow–originated antigen presenting cells derived from monocyte-macrophages DC precursors. Intestinal DCs are found within organized lymphoid tissue such as Peyer's patches, mesenteric lymph nodes, and in the lamina propria (LP). DCs tolerate antigens and gut symbionts while strategizing adaptive immune responses to combat pathogens. In the LP, myeloid DCs can be classified into 2 different subsets: plasmacytoid DCs and conventional DCs (cDCs). CDCs in the gut can be subdivided into the CD103+CD11b− cDC1, and the CD103+CD11b+ and CD103−CD11b+ cDC2 subsets.3 The development of these conventional DCs subsets is under the regulation of distinct transcription factors. cDC1 development is defined by basic leucine zipper transcription factor ATF-4 like 3 (BATF3) and interferon regulatory factor 8 (IRF8), while cDC2 development is regulated by IRF4.3 In the mesenteric lymph nodes, cDC1s classified as CD8α+CD11b−, present luminal antigens to T cells. cDC1s can induce T helper type 1 (Th1) responses, T regulatory cells to promote tolerance, and efficiently cross-present antigens to cytotoxic CD8+ T cells to enhance immunity against bacteria, viruses, and parasites, and induce antitumor immunity.3 cDC2s are more heterogenous than cDC1s and function in supporting Th responses (including Th1, Th2, or Th17), inducing IgA, and stimulating the differentiation of T regulatory cells. Through IL-23 secretion, cDC2s can also activate innate lymphoid cells 3 to secrete IL-22.4 Hence, DCs control immunity versus tolerance influencing gut function. However, the implications of chronic alcohol misuse on intestinal DC subsets and their role in ALD pathogenesis remain elusive.3 In this issue of Hepatology, Hao and colleagues comprehensively elucidate the protective role of Batf3-dependent cDC1s on ethanol-induced intestinal permeability, bacterial endotoxin translocation, and liver injury, as well as the involvement of the microbiome, particularly Akkermansia muciniphila as the mechanism preventing ethanol-induced liver disease (Figure 1).5 For this purpose, the authors used a modified NIAAA model of ethanol-induced liver disease, where male mice were fed with the Lieber DeCarli liquid diet containing alcohol or isocaloric maltose dextrin control for 8 weeks with a gradual increase of ethanol concentration, followed by a single dose of ethanol at 4 g/kg or isocaloric maltose dextrin 4 hours before tissue collection. The authors demonstrated that chronic alcohol feeding increased the total number of conventional DCs in Peyer's pathes, and in the small and large intestinal LP. However, the numbers of Batf3-dependent CD103+CD11b− cDC1s were markedly decreased and secreted less ileal IL-12 protein levels. IL-12, a proinflammatory cytokine, mediates the induction of interferon gamma (IFN-γ)-secreting Th1 and CD8 T cells.6 Despite IFN-γ proinflammatory properties, IFN-γ-inducible genes such as indoleamine 2,3 dioxygenase (IDO1) can also promote an anti-inflammatory response.6 The authors found that ethanol-mediated cDC1 reduction resulted in a consequent decline of ileal Th1 and CD8+IFN-γ+ T-cell proportions in conjunction with a decrease of ileal IFN-γ protein and mRNA levels. Consequently, mRNA levels of downstream targets such as ileal C-X-C motif chemokine ligand 9 (Cxcl9), Ido, interferon gamma-induced protein 10 (Ip-10), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) protein levels were downregulated by ethanol exposure.FIGURE 1: cDC1s prevent ethanol-induced liver disease through the maintenance of intestinal Akkermansia muciniphila. CD103+ CD101b− cDC1s are a gatekeeper for ethanol-induced liver disease in mice through regulation of IL-12-IFN-γ-STAT3-AMPs signaling that results in the maintenance of intestinal A. muciniphila, which in turn prevents ethanol-induced tight junction disruption. Ethanol alters Th1 and CD8 T cell, responses resulting in a reduction of A. muciniphila and disruption of tight junctions allowing bacterial translocation. In the liver, translocated microbial products promote inflammation and accumulation of M1, macrophages, and neutrophils, triggering inflammation and steatosis. This figure was created using Biorender.com. Abbreviations: AMP, antimicrobial peptides; Batf3, basic leucine zipper transcription factor ATF-4 like 3; CXCL, C-X-C Motif Chemokine Ligand; Defa5, defensin alpha 5; LPS, lipopolysaccharides; Reg3, regenerating islet-derived protein; STAT3, signal transducer and activator of transcription 3; Th1, T helper type 1.In the immune system, Th1 cells are a subtype of CD4+ T cells that participate in the defense against intracellular parasites and are related to autoimmune responses through inflammatory cell activation by means of IFN-γ. In the same line as Hao and colleagues work, studies have found that alcohol misuse decreases IL-12 and IFN-γ in the serum, mesenteric lymph nodes, and spleen. However, there are reports on the increased levels of Th1 cytokine IL-12, after lipopolysaccharide insult, in the liver of ethanol-fed mice.4 The elevated serum levels of IL-12 were also present in alcohol use disorder patients.7 However, as discussed by the authors, intestinal IL-12 participates in Th1 and CD8 T-cell adaptive immune responses, which are vital for antimicrobial peptide (AMP)s secretion. IFN-γ, stimulated by IL-12, offers immune protection against intestinal pathogens and plays a critical role in the intestine by regulating STAT-AMPs secretion. Chronic ethanol exposure leads to the downregulation of IFN-γ.8 IFN-γ participates in colitis, Paneth cell death and regeneration, and intestinal permeability.9 The role of the intestinal Th1/IL-12/IFN-γ axis and its implications in intestinal physiology in ethanol-induced liver injury was elusive until this issue was addressed in the study by Hao and colleagues. In doing so, the authors used Batf3−/− mice as a genetic mouse model with cDC1 deletion to determine the role of cDC1s in mediating ethanol-induced liver injury. The authors demonstrated that depletion of IL-12–secreting cDC1s in Batf3−/− mice dramatically inhibited ileal Th1 and CD8 T-cell adaptive immune responses, leading to an exacerbated ethanol-induced steatohepatitis and endoplasmic reticulum stress. Notably, the lack of cDC1s aggravated ethanol-induced intestinal tight junction disruption, resulting in an increased pathogen-associated molecular patterns translocation to the circulation and liver in ethanol-fed Batf3−/− mice. Accumulation of pathogen-associated molecular patterns in the liver was associated with an increase in the frequencies of Ly6g+Cd11b+ neutrophils, a higher number of CD80+CD126+F4/80+ M1 macrophages, and increased proportions of infiltrated monocytes in the liver. Interestingly, the authors found that adoptive transfer of MHCII+CD11c+CD103+Clec9A+ cDC1 restored ileal IL-12 protein levels, frequencies of Th1 and CD8+TIFN-γ+ cells, ileal IFN-γ+ protein levels, ileal levels of p-STAT3 and AMP secretion, as well as prevented ethanol-mediated intestinal barrier disruption and pathogen-associated molecular patterns translocations to the liver. These responses resulted in diminished frequencies of neutrophils and numbers of infiltrated M1 macrophages in the liver and alleviated ethanol-induced steatohepatitis. Hence, this approach shed light on the role of the intestinal cDC1/Th1/IL-12/ IFN-γ axis in intestinal physiopathology in the context of ethanol-induced liver disease. The cDC1s also play an important role in tolerance development; however, how deficiency of cDC1s and tolerogenic responses influence the intestinal barrier function and bacterial translocation in ethanol-induced liver injury is poorly characterized. On a mechanistic level, Batf3-dependent ablation of cDC1s would prevent the differentiation of T regulatory cells.10 As shown and discussed by the authors, cDC2s are increased in Batf3−/− mice along with an induced Th17 response. cDC2s are also important in maintaining tolerance and could switch to proinflammatory Th17 responses due to cDC1 deficiency. Recent studies have shown the importance of cDC1s in inducing a metabolic switch on cDC2s from inflammatory to tolerogenic responses through IDO1.11 Therefore, the role of cDC2s on disease or health might depend on their ability to regulate tolerance versus inflammatory responses. In addition, cDC2s and cDC1s regulate IgA synthesis in the gut.10 Given the relevance of IgA on mucosal barrier protection, whether IgA is relevant in this ethanol-induced liver injury model should also be considered. Lastly, the implications of cDC1s in mediating ethanol-induced liver injury may also depend on the anatomic distribution in the intestine. The ratio of cDC1s versus cDC2s changes along the intestine. cDC2 abundance is pronounced in the small intestinal LP and rare in the colonic LP, while cDC1s are the major subset in the colonic LP. Hao and colleagues' work focused on the cDC1s in the ileum. Given the anatomic zonation of cDC1s, and the importance of adaptive immune responses in shaping the microbial composition, it would be reasonable to study adaptive immune responses, molecular features, and the variance of microbes along the gut. The authors in previous studies reported that ethanol-mediated inhibition of intestinal IFN-γ signaling and consequent reduction of AMPs secretion reduce cecal A. muciniphila abundance, which results in gut barrier disruption and liver injury.8 These findings were consistent with a reduction of ileal mRNA levels of the AMPs defensin alpha 5 (Defa5), regenerating islet-derived protein 3β (Reg3β) and Reg3γ, and a consequent cecal A. muciniphila reduction in ethanol-fed Batf3−/− mice. Extensive work on the relevance of AMPs in ethanol-induced liver disease has been published.12 To evaluate the role of IFN-γ and IL-12 on AMPs and A. muciniphila abundance, the authors administered IFN-γ or IL-12 twice a week through i.p. injection for the last 4 weeks. Administration of IFN-γ (300 ng/mouse) reversed ethanol-mediated inhibition of ileal p-STAT3 and AMPs levels and restored the abundance of A. muciniphila in ethanol-fed WT and Batf3−/− mice. Similarly, treatment with IL-12 (5 μg/mouse) reversed ethanol and Batf3 ablation-mediated reduction of ileal Ifn-γ, Ido, and Ip-10 expression, as well as ileal p-STAT3 protein levels and ileal Defa5, Reg3β, and Reg3γ mRNA levels that resulted in an elevated abundance of A. muciniphila. Knowing the relevance of Th1 in protecting against pathogens, the authors observed higher numbers of known pathogens associated with ALD such as Enterococcus in Batf3−/− mice. To understand the role of A. muciniphila in cDC1-deficient Batf3−/− mice, the authors orally administered 5×108 CFU of A. muciniphila every other day for the last 4 weeks. A. muciniphila administration ameliorated Batf3-mediated effects such as intestinal epithelial tight junction disruption and pathogen-associated molecular patterns translocation and alleviated from ethanol-induced steatohepatitis and endoplasmic reticulum stress. However, A. muciniphila administration did not influence AMPs expression and did not alter cDC1 numbers. Therefore, cDC1s confer ethanol-induced liver injury protection through the protective effects of A. muciniphila on the gastrointestinal barrier and AMPs secretion on antibacterial response and bacterial translocation. Lastly, the authors used Lactobacillus reuteri as a known probiotic to induce cDC1s that skew T cells toward Th1 polarization by means of IL-12 signaling. Administration of 1.5 ×109 CFU L. reuteri every other day for the last 4 weeks reversed the inhibitory effect of ethanol on the number of cDC1s, IL-12 secretion, on the number of Th1 and IFN-γ+ CD8+ T cells, as well as IFN-γ protein levels and consequently restored AMPs levels, induced growth of A. muciniphila, and prevented pathogen-associated molecular patterns translocation to the liver. Methodologically, it should be cautioned that the administration of high doses of ethanol 4 hours before immune cell isolation could result in acute and specific inflammatory responses. Although DC purification with magnetic beads and sorting for DC enrichment are excellent techniques to study DCs, there is a possibility that such methods could activate DCs and may have an impact on the results of the study. Taken together, the study of Hao and colleagues demonstrated the interaction between intestinal immune cells and microbiota in mediating ethanol-induced liver injury. Intestinal cDC1s are gatekeepers in preventing ethanol-induced liver disease through the regulation of IL-12-IFN-γ-STAT3-AMPs signaling and maintenance of intestinal A. muciniphila. This study implies that intestinal cDC1s could be a therapeutic target for the treatment of ethanol-induced liver injury. The study by Hao and colleagues provides us with a new avenue to explore targeted therapies based on LP-DCs, adaptive immune responses, and microbiome-liver axis in patients with ALD. Several clinical trials targeting the microbiome-immune system interaction in ALD patients have been proposed with the use of probiotics, fecal microbiota transplantation, and antibiotics to restore the intestinal homeostasis.4 Future studies should also focus on other intestinal immune cells in the LP and how changes in the LP-immune response alter the abundance and composition of the microbiota, the metagenome, and the metabolome in ethanol-induced liver injury. The results will pave a way for the discovery of novel therapeutic strategies for ALD.